Vascularized gastric cancer organ chip and preparation method thereof

Abstract

The invention relates to the technical field of tumor biomedical engineering and organ chips, in particular to a vascularized gastric cancer organ chip and a preparation method thereof.The chip is composed of an integrated micro-fluidic main body, an annular micro-column array and an optical sealing film, and a central culture cavity is divided into a tumor area and a blood vessel area by micro-columns; the preparation method comprises the following steps: injecting a fibrous protein solution containing human umbilical vein endothelial cells and cancer-related fibroblasts into a vascular region, and adding thrombin for in-situ gelation to form a pre-vascularized network; the method comprises the following steps: mixing a patient-derived gastric cancer organ with a methacrylic acid esterified gelatin pre-polymerized solution, injecting the mixture into a tumor area, and carrying out photo-crosslinking immobilization; culturing for 7-14 days under the dynamic perfusion condition of a mixed culture medium to obtain a three-dimensional gastric cancer organ model containing a capillary network; the method is expected to be used for rapid, low-cost and high-throughput screening of chemotherapy or anti-angiogenesis drugs and accurate prediction of individual curative effects of patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of tumor bioengineering and organ chip technology, in particular to a vascularized gastric cancer organoid chip and a preparation method thereof. BACKGROUND

[0002] In recent years, patient-derived tumor organoids (PDTOs) have become a research hotspot for in vitro models of gastric cancer. This technology can reconstruct highly faithful and amplifiable miniature tumor tissues in a three-dimensional matrix. Preclinical studies have shown that PDTOs can better predict individual patient responses to conventional chemotherapy regimens. However, traditional PDTO drug sensitivity evaluation still faces key limitations: due to the lack of blood vessels, stroma, and shear flow, it only reflects the effects of drugs under diffusion feeding conditions, making it difficult to accurately deduce drug delivery and drug resistance dynamics in solid tumors.

[0003] Angiogenesis is the core driving force for the growth, infiltration, and distant metastasis of gastric cancer. A 3D model without blood vessels cannot reproduce the nutrient supply gradient, endothelial-tumor signal interaction, and drug transvascular barrier process, resulting in limited in vitro-in vivo conversion rates. A large real-world study by Yang Jinji's team on lung cancer also pointed out that the prediction of chemotherapy sensitivity based on avascular PDTOs still has significant deviations from the actual efficacy of patients, further highlighting the importance of the peripheral vascular microenvironment.

[0004] Organ-chip technology provides a breakthrough for this problem. Using microfluidic platforms, it can precisely construct and perfuse vascular endothelial networks at the micron scale, enabling co-culture of tumor organoids with fibroblasts, immune cells, and other components, while dynamically regulating flow rate, shear force, and concentration gradient. This not only improves the simulation of drug penetration and metabolism in real conditions, but also supports high-throughput parallel testing, providing a more clinically relevant evaluation system for individual chemotherapy regimens.

[0005] Although various vascularized organoid chip prototypes have emerged in recent years for pancreatic cancer, breast cancer, and other tumors, there are still few mature reports in the field of gastric cancer. Most existing models use cell lines rather than patient-derived cells, and the efficiency and stability of the vascular network construction are insufficient, making it difficult to truly reproduce the unique blood vessel-stromal interaction of gastric cancer. SUMMARY

[0006] Based on the above problems, the present application proposes a vascularized gastric cancer organoid chip and a preparation method thereof. Endothelial cells and tumor-associated fibroblasts form a vascularized environment and co-culture with gastric cancer organoids to construct a gastric cancer vascularized microenvironment model.

[0007] The application provides a vascularized gastric cancer organoid chip, which comprises a microfluidic chip body with a central culture chamber, wherein a microcolumn array arranged in a ring shape is arranged in the culture chamber, and the culture chamber is divided into a concentric inner region and an outer region; the chip body is further provided with at least one first sample adding channel for introducing a cell suspension into the outer region, at least one second sample adding channel for introducing an organoid into the inner region, and a culture medium storage tank connected to the culture chamber; wherein the inner region contains a patient-derived gastric cancer organoid, and the outer region is filled with a three-dimensional hydrogel matrix containing endothelial cells and tumor-associated fibroblasts to form a co-culture structure of a blood vessel network and a gastric cancer organoid in the culture chamber.

[0008] The microcolumn array is arranged in the central culture chamber in a ring shape and uniformly distributed, the microcolumn diameter is 50-200 μm, and the interval between adjacent microcolumns is 50-300 μm, which is used to separate the culture chamber into the concentric inner region and outer region by surface tension.

[0009] The inner region three-dimensional matrix system is filled with methacrylated gelatin (GelMA) or collagen hydrogel and fixed with a patient-derived gastric cancer organoid.

[0010] The outer region angiogenesis system is filled with a fibrin hydrogel containing endothelial cells and tumor-associated fibroblasts, which is used to construct a perfused capillary network.

[0011] The fluid interface module is at least one pair of first and second sample adding channels respectively connected to the outer region and the inner region, and four culture medium storage tanks connected to the culture chamber through microfluid channels, which are used to supply culture medium and drug solution.

[0012] The packaging assembly covers the optical pressure-sensitive film on the top of the chip to ensure aseptic sealing and compatibility with high-magnification microscopic imaging.

[0013] Further, the patient-derived gastric cancer organoid is obtained by in vitro culture of tumor tissue of a gastric cancer patient, the tumor-associated fibroblasts are primary fibroblasts separated from the tumor stroma of the patient, and the endothelial cells are human microvascular endothelial cells or human umbilical vein endothelial cells.

[0014] Further, the microcolumn diameter of the microcolumn array is 50-200 μm, the interval between adjacent microcolumns is 50-300 μm, and the microcolumn array is uniformly distributed in a ring shape, which is used to isolate the culture solution or gel in the inner region and the outer region from each other by surface tension.

[0015] Further, the three-dimensional hydrogel matrix comprises a fibrin hydrogel and / or a methacrylated gelatin hydrogel, wherein the outer region matrix is a fibrin gel for supporting the three-dimensional growth of the endothelial cells and fibroblasts; the inner region matrix is a methacrylated gelatin or collagen gel for fixing and supporting the gastric cancer organoids.

[0016] Further, the chip body is made of a biocompatible transparent resin material, is integrally formed by a digital light processing (DLP) 3D printing method, and has a transparent PCR optical adhesive film attached to the surface of the chip body to seal the culture chamber; the number of culture medium reservoirs is four, which are distributed around the culture chamber to provide storage and flow supply of the culture medium.

[0017] The application also provides a preparation method of a vascularized gastric cancer organoid chip, comprising the following steps:

[0018] S1, the chip body is formed by 3D printing to form the culture chamber, the microcolumn array, the sample adding channel and the reservoir structure, and a transparent sealing film is attached to the surface of the chip body to seal the culture chamber, and only openings are reserved at the reservoir and the sample adding channel;

[0019] S2, the endothelial cells and tumor-related fibroblasts are suspended in a coagulable hydrogel precursor solution, injected into the outer region of the chip culture chamber through the first sample adding channel, and the precursor solution is induced to coagulate to form a three-dimensional matrix gel containing the cells;

[0020] S3, the chip treated in step (2) is subjected to cell culture, the endothelial cells are extended and connected in the matrix gel to form a pre-vascularized capillary network;

[0021] S4, the patient-derived gastric cancer organoids are added to the inner region of the culture chamber, and the organoids are fixed in the inner region by using the hydrogel matrix;

[0022] S5, the culture is continued, and the organoids are co-cultured with the capillary network for a period of time to obtain a gastric cancer organoid model containing a vascular network.

[0023] Further, the 3D printing in step (1) adopts a digital light processing (DLP) technology, and the layer thickness is preferably 50-100 μm to form a fine microcolumn array structure; after the printing is completed, the chip body is subjected to residual cleaning and ultraviolet disinfection, and the transparent sealing film is a polymer film adhered by pressure-sensitive adhesive, so that the chip forms a closed microfluidic culture environment.

[0024] Further, the hydrogel precursor solution in step (2) is a solution containing 2.5-5 mg / mL fibrinogen, and the induced coagulation is achieved by adding thrombin; in step (4), the gastric cancer organoid is mixed with 50-100 mg / mL methacrylated gelatin pre-polymer solution before being injected into the inner region, and is cross-linked into a gel by ultraviolet light irradiation for 5-30 seconds, so as to fix the organoid in the inner region.

[0025] Compared with the prior art, the present application has the following beneficial effects:

[0026] (1) The patient-derived gastric cancer organoid and cancer-associated fibroblasts are co-cultured, which is closer to the real biological characteristics of tumor tissues than the traditional model using a single tumor cell line, and improves the accuracy of the model in predicting drug responses.

[0027] (2) The biological hydrogel such as methacrylated gelatin (GelMA) is used to replace the commercial basement membrane extract (Matrigel) as a three-dimensional culture scaffold, which has the advantages of clear composition, stable batch, and high reproducibility, and avoids the experimental errors caused by the uncertain composition of traditional matrix glue.

[0028] (3) The 3D printing technology of digital light processing (DLP) is used to directly manufacture the microfluidic chip, which greatly simplifies the process flow, reduces the manufacturing cost and shortens the production cycle compared with the traditional photolithography-soft lithography printing method, and makes the chip design more flexible and can be quickly iterated and optimized. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 is a schematic diagram of the overall structure of the gastric cancer vascularized organoid chip of the present application.

[0030] Figure 2 is a split schematic diagram of the chip assembly of the present application.

[0031] Figure 3 is a planar layout diagram of the chip of the present application.

[0032] Figure 4 is a cross-sectional view of the chip A-A of the present application.

[0033] Figure 5 is a flowchart of the preparation of the gastric cancer vascularized organoid chip of the present application.

[0034] Figure 6Figure 1 is a microscope photo of the chip co-culture forming a vascularized organoid model of the present application. Wherein (a) is a gastric adenocarcinoma organoid (GCOs) and tumor-associated fibroblasts (CAFs) extracted from a gastric cancer patient's surgical resection tissue, scale 200 μm; (b) is a culture endothelial cell (HUVEC-GFP) tube formation effect diagram, endothelial cells can grow significant new vascular sprouts to the center within 1-3 days, scale 500 μm; (c) is a schematic diagram of HUVEC-GFP and gastric cancer organoid co-culture, scale 500 μm.

[0035] Wherein, 1, chip body; 2, PCR optical adhesive pressure sensitive film; 3, sample well; 4, micro column; 5, culture medium reservoir. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application.

[0037] Embodiment 1

[0038] The present embodiment provides a method for manufacturing a microfluidic chip, comprising the following steps:

[0039] First, design the chip structure by three-dimensional modeling software.

[0040] The chip body 1 has a size of about 30 mm in length, 20 mm in width and 5 mm in height, and the middle part is a culture chamber. The culture chamber has a diameter of about 10 mm, the bottom is integrally formed with the chip body 1, and the top is open. The chamber is uniformly provided with micro columns with a size of 300 μm in length, 200 μm in width and 500 μm in height and a spacing of 300 μm, which divide the chamber into an inner ring area (2 mm in diameter) and an outer ring area (5 mm in diameter).

[0041] Four cylindrical reservoirs 5 with a diameter of 5 mm and a height of 5 mm are also designed on the chip, which are respectively located at the four corners of the culture chamber and are connected with the culture chamber through fine channels, for storing culture medium and supplying the culture chamber with culture medium through capillary force. In addition, sample wells with a diameter of 1 mm are provided on both sides of the culture chamber, one side of the sample well is connected with the outer ring area of the culture chamber through a micro channel (for adding endothelial / fibroblast cell suspension), and the other side of the sample well is connected with the inner ring area of the culture chamber (for adding organoid suspension). This design ensures that the different cell sample channels do not interfere with each other, and also enables the culture medium to flow and circulate in the entire chip.

[0042] After the design is completed, a DLP light-cured 3D printer is used to print the above chip structure.

[0043] The printing material uses biocompatible photosensitive resin, and in this embodiment, the biocompatible transparent resin of Shenzhen Cubic Technology Co., Ltd. is used, and the layer thickness is set to 50 μm to ensure the forming precision of the micro column 4 and other fine structures.

[0044] During the printing process, the model is solidified layer by layer, and the solid chip is obtained after about half an hour.

[0045] After printing, the chip is taken off the printing platform and sequentially post-processed: first, ultrasonic cleaning in alcohol for 3 minutes to remove un-solidified resin residues, then post-curing for 60 minutes, and then standing and soaking in a container containing deionized water for 24 hours to further dissolve organic solvents and non-polymeric substances.

[0046] Next, the chip is soaked in a 75% ethanol solution for 30 minutes, and at the same time, it is irradiated with a UV lamp for 15 minutes to achieve the purpose of disinfection and sterilization.

[0047] Finally, the chip is taken out and dried in a sterile clean bench for standby.

[0048] The obtained chip body 1 is transparent and smooth, and the micro column array and micro channel structure are clearly visible, and the size precision is consistent with the design value.

[0049] Subsequently, a pre-cut PCR sealing plate optical film 2 (a transparent polymer film with a thickness of about 100 μm and adhesive) is carefully covered and pasted on the top surface of the chip body 1, ensuring that the film tightly seals the culture cavity and micro channel, and only leaving openings at the reservoir and sample well positions.

[0050] This step forms a closed microfluidic chip device while retaining the necessary openings for subsequent introduction of cells and culture medium.

[0051] The chip prepared in this embodiment is rinsed with sterile PBS before use to ensure that there is no residual disinfectant affecting the cells.

[0052] Example 2

[0053] This embodiment introduces the preparation method of gastric cancer organoids, which can be used for subsequent chip experiments, including the following steps:

[0054] Fresh tumor tissue of gastric adenocarcinoma patients (surgical resection specimens obtained with the patient's informed consent) is sent to the laboratory after adding 2x antibiotics in ice-cold PBS. The tumor tissue is cut into small pieces of about 1 mm^3, and digested with digestive enzyme solution at 37°C for 1 hour, and gently mixed every 15 minutes.

[0055] The digestive enzyme solution in this embodiment is a mixture of 1 mg / mL collagenase type IV, 0.5 mg / mL hyaluronidase, and DNase I.

[0056] After digestion, the digestion was terminated with culture medium and filtered through a 40 pm cell strainer, and the flow-through was collected and centrifuged to obtain a cell pellet. The cell pellet was resuspended in chilled Matrigel matrix gel, and was dispensed in 50 pL droplets on a culture plate and incubated at 37 °C for 10 min to solidify into a spherical gel. Subsequently, gastric cancer organoid culture medium was added to cover the gel, and the medium was replaced 2-3 times per week.

[0057] In addition, the Matrigel matrix gel can be replaced with a 5% GelMA solution containing a photoinitiator 0.25% LAP;

[0058] The organoid culture medium includes advanced DMEM / F12, B27, N2, EGF, Noggin, R-spondinl, Wnt3a conditioned medium, Gastrin, Nicotinamide, A83-01, Y-27632.

[0059] After about 1-2 weeks, translucent spherical organoid structures with a diameter of 50-200 pm were observed in the Matrigel gel under a microscope.

[0060] Example 3

[0061] In this example, the microfluidic chip prepared in Example 1 was combined with the organoid application obtained in Example 2 to construct a gastric cancer vascularized organoid model, and the specific steps are as follows.

[0062] First, human umbilical vein endothelial cells (HUVEC) were purchased from a cell bank and cultured in EGM-2 medium containing growth factors such as VEGF and bFGF at 37 °C and 5% CO2 to expand to the logarithmic growth phase for use.

[0063] At the same time, tumor-associated fibroblasts (CAF) were isolated from fresh tumor tissue resected from a gastric cancer patient: the tissue block was digested with a mixed enzyme solution of collagenase-IV and hyaluronidase, filtered and centrifuged, and then placed in a culture dish to separate using the selective adhesion properties of fibroblasts, and then cultured in DMEM / F12 medium containing 20% fetal bovine serum and subcultured to the 3rd passage or more for use.

[0064] Before use, the HUVEC were fluorescently labeled by transfecting a GFP expression vector using a lentivirus to obtain stable green fluorescent expression, which facilitated subsequent dynamic imaging of the vascular network; the CAF remained unlabeled to avoid signal overlap.

[0065] Finally, the HUVEC and CAF were mixed at a cell number ratio of 3: 1, and the total cell concentration was adjusted to about 8 x 106 cells / mL, and stored in an ice bath for temporary storage, and used as needed to ensure cell activity and subsequent loading efficiency.

[0066] Prepare a 10 mg mL-1 sterile fibrinogen solution and a 100 U mL-1 thrombin solution; fibrinogen can rapidly polymerize into a three-dimensional fibrin network upon encountering thrombin, providing an in vivo scaffold for endothelial / fibroblast cells, with final concentrations of 5 mg mL-1 and 2 U mL-1, respectively;

[0067] Take the sterilized and air-dried chip and place it on an ice plate to avoid partial solidification before being filled.

[0068] Mix HUVEC / CAF cells at a ratio of 3:1 and resuspend them in 10 μL of the fibrinogen solution, and mix with the thrombin solution, blow 5-6 times, and quickly inject through the first sample well of the chip.

[0069] Stop the operation and let it stand for 30 s, and the fibrinogen gels in situ in the presence of cells, filling the outer ring culture area. The microcolumn array can generate a capillary barrier to block the overflow of the gel; at this time, the outer ring is observed to be translucent, and the inner ring remains blank, indicating good isolation effect.

[0070] Move the chip into a 37°C incubator and let it stand for 15 min to allow the fibrin network to completely solidify and begin to support angiogenesis.

[0071] To achieve uniform distribution, suspend the pretreated gastric cancer organoids (100-200 μm in diameter) in 50 mg mL-1 methacrylated gelatin (GelMA) pre-polymer solution and maintain at 37°C to maintain fluidity.

[0072] Inject 5 μL of the suspension into the inner ring through sample well 3; the GelMA fluid can fully infiltrate the cavity surrounded by the microcolumn.

[0073] Use a handheld 405 nm LED light source to irradiate for 20 s to trigger the photocrosslinking reaction, quickly fixing the organoids in the central region to prevent displacement caused by subsequent perfusion flushing.

[0074] Inject 37°C preheated co-culture medium (EGM-2:PDO medium = 1:1, supplemented with 5% FBS + 50 ng mL-1 VEGF) into the four reservoirs. Until the liquid level is level with sample well 3, ensure that the convection is balanced.

[0075] Gently shake the chip to remove residual air bubbles to prevent them from lodging and disrupting the flow path and affecting cell nourishment.

[0076] Place the chip in a 37°C, 5% CO2 incubator to begin dynamic co-culture of the vascular network and gastric cancer organoids, and replace the medium in the reservoirs with fresh medium every 24 hours. In addition, collect the supernatant at the same time for analysis.

[0077] In the first 48 hours of co-culture, mainly the formation stage of the vascular network.

[0078] The chip can be taken out at 24 and 48 hours of culture, and the morphology of endothelial cells can be observed by inverted fluorescence microscope: at 24 hours, endothelial cells can be seen to form short chain-like structures along the fibrin gel; after 48 hours, most endothelial cells have connected into a network, and an obvious capillary-like network structure has been formed in the gel, while CAF cells have elongated and are distributed together with endothelial cells at the network nodes, which indicates that the pre-vascularization process has been successfully completed.

[0079] Thereafter, the co-culture stage of the organoid and the vascular network begins on day 3.

[0080] On day 1 after the addition of the organoid, it is observed that some organoids adhere to the surface of the micro-pillars or the gel interface, and maintain a spherical shape with no obvious change in diameter; the vascular network continues to expand and gradually surrounds the organoid.

[0081] On day 7, a stable organoid-vascular co-culture system has been formed in the chip: the green fluorescently labeled vascular network almost covers the entire outer ring area, and capillary-like filaments extend to the location near the organoid in the inner ring area; the organoid itself maintains good vitality, and some have slightly increased in size and developed complex gland lumen structures (the light and dark structures inside the organoid can be seen under a microscope, indicating that it has formed a gland lumen).

[0082] The above describes the present application and its embodiments, which are not limiting, and the embodiments shown in the drawings are only one of the embodiments of the present application, and the actual structure is not limited thereto. In summary, if a person of ordinary skill in the art is inspired thereby, without departing from the spirit of the present application, without creative design, similar structural modes and embodiments of the technical solutions can be designed, which should all belong to the protection scope of the present application.

Claims

1. A vascularized gastric cancer organoid microarray, characterized in that, include: The microfluidic chip body has a central culture chamber, in which a ring-shaped array of micropillars is arranged to divide the culture chamber into concentric inner and outer regions; The chip body also has at least one first sample dispensing channel for introducing cell suspension into the outer region, at least one second sample dispensing channel for introducing organoids into the inner region, and a culture medium reservoir communicating with the culture chamber. The inner region contains patient-derived gastric cancer organoids, and the outer region is filled with a three-dimensional hydrogel matrix containing endothelial cells and tumor-associated fibroblasts to form a co-culture structure of a vascular network and gastric cancer organoids within the culture chamber. The micropillar array, located in the central culture chamber, is uniformly distributed in a ring shape and is used to divide the culture chamber into concentric inner and outer regions by means of surface tension; The inner three-dimensional matrix system is filled with methacrylated gelatin or collagen hydrogel and fixes patient-derived gastric cancer organoids; the outer angiogenesis system is filled with fibrin hydrogel containing endothelial cells and tumor-associated fibroblasts to construct a perfusion capillary network. The sample addition channels are connected to the outer and inner zones respectively, and the four culture medium storage tanks are connected to the culture chamber through microchannels to supply culture medium and drug solutions; An optical pressure-sensitive film covering the top of the chip ensures sterile sealing and compatibility with high-magnification microscopy.

2. The vascularized gastric cancer organoid chip according to claim 1, characterized in that, The patient-derived gastric cancer organoids are obtained by in vitro culture of tumor tissue from gastric cancer patients. The tumor-associated fibroblasts are primary fibroblasts isolated from the tumor matrix of the patients. The endothelial cells are human microvascular endothelial cells or human umbilical vein endothelial cells.

3. The vascularized gastric cancer organoid chip according to claim 1, characterized in that, The micropillars in the micropillar array have a diameter of 50–200 μm and a spacing of 50–300 μm between adjacent micropillars. The micropillar array is uniformly distributed in a ring shape and is used to isolate the culture medium or gel in the inner and outer regions from each other by utilizing surface tension.

4. The vascularized gastric cancer organoid chip according to claim 1, characterized in that, The three-dimensional hydrogel matrix includes fibrin hydrogel and / or methacrylated gelatin hydrogel, wherein the outer region matrix is ​​fibrin gel to support the three-dimensional growth of the endothelial cells and fibroblasts; and the inner region matrix is ​​methacrylated gelatin or collagen gel to fix and support the gastric cancer organoids.

5. The vascularized gastric cancer organoid chip according to claim 1, characterized in that, The chip body is made of biocompatible transparent resin material and is integrally formed by digital light processing 3D printing. A transparent PCR optical film is attached to the surface of the chip body to seal the culture chamber. There are four culture medium storage tanks distributed around the culture chamber to provide storage and flow supply of culture medium.

6. A method for preparing a vascularized gastric cancer organoid microarray according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Provide the chip body as described in claim 1, and use 3D printing to form the culture chamber, micropillar array, sample dispensing channel and liquid storage tank structure, and attach a transparent sealing film to the surface of the chip body to seal the culture chamber, leaving openings only at the liquid storage tank and sample dispensing channel; S2. Endothelial cells and tumor-associated fibroblasts are suspended in a solidifiable hydrogel precursor solution and injected into the outer region of the chip culture chamber through the first sample delivery channel to induce the precursor solution to solidify and form a three-dimensional matrix gel containing the cells. S3. The chip after step (2) is subjected to cell culture, so that the endothelial cells extend and connect in the matrix gel to form a prevascularized capillary network. S4. Add the gastric cancer organoids from the patient to the inner region of the culture chamber, and fix the organoids to the inner region using a hydrogel matrix; S5. Continue culturing, co-culturing the organoids with the capillary network for a period of time to obtain a gastric cancer organoid model containing a vascular network.

7. The method according to claim 6, characterized in that, The 3D printing in step (1) uses digital light processing (DLP) technology, and the layer thickness is preferably 50–100 μm to form the fine micropillar array structure; After printing, the chip body is cleaned to remove residue and disinfected with ultraviolet light. The transparent sealing film is a polymer film adhered with pressure-sensitive adhesive, which makes the chip form a closed microfluidic culture environment.

8. The method according to claim 6, characterized in that, The hydrogel precursor solution in step (2) is a solution containing 2.5-5 mg / mL fibrinogen, and the induction of coagulation is achieved by adding thrombin; in step (4), the gastric cancer organoids are mixed with 50-100 mg / mL of methacrylated gelatin prepolymer solution and injected into the inner region, and then irradiated with ultraviolet light for 5-30 seconds to crosslink them into a gel, thereby fixing the organoids in the inner region.

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